FIG. 5.

The complex recruited to the promoter by CRG contains components of an RNA polymerase II complex, as indicated by an antibody supershift assay. (A) The method uses rabbit primary antibodies specific for components of the polymerase complex, a biotinylated goat anti-rabbit secondary antibody, and streptavidin-conjugated Dynabeads to bind the complexes into larger “supercomplexes.” (B) Binding reactions contained primary antibody (1° Ab) directed against CREB, secondary antibody (2° Ab), and/or beads, as indicated, with 5 fmol of probe, 10 fmol of purified CRG, and 3 μg of RLNE. (C) Primary antibodies specific for CREB, TFIIB, the large subunit of RNA polymerase II, TBP, or an irrelevant T7 antibody were included with secondary antibody and streptavidin beads where indicated, as in panel B.